Vector borne viruses from different virus families account for many medically significant viral pathogens. More specifically, the vector borne flaviviruses, which belong to the Family Flaviviridae, genus Flavivirus, comprise some of the most important emerging and re-emerging viral pathogens. The tick borne flaviviruses (TBFV) include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Alkhurma hemorrhagic fever virus, Powassan/deer tick virus (POWV/DTV) and Langat virus (LGTV). TBFV are generally transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. The mosquito borne flaviviruses include West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). The dramatic and ongoing pandemic attributed to the MBFV Zika virus is a topic of considerable interest and significance because of its capacity to cause severe neurological defects including severe microcephaly and even fetal wastage in human fetuses. The public health implications of ZIKV are troubling. Our current research is focused on the TBFV, but studying the biology of TBFV will elucidate the biology of other vector borne viruses. The research in our laboratory employs virology, immunology, advanced imaging techniques, genomics, cell biology, molecular biology, and vector biology. We primarily study LGTV, a naturally attenuated member of the TBFV that shares approximately 80% identity with TBEV at the amino acid level. LGTV can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. In addition, in the past year we are also studying the more virulent autochthonous BSL-3 POWV/DTV. Neither of these 2 agents are Select Agents which greatly facilitate research studies. In addition, we have also begun to study the BSL2 MBFV, Zika virus. With the recent emergence of Zika in Latin America and the US, similar avenues of inquiry are being explored for that vexatious pathogen. Comparison of VBFV cytoarchitecture in mammalian and tick cells. A key difference between TBFV infection of vertebrate and arthropod host systems is that infection of ticks is persistent and non-cytolytic, whereas infection of mammalian hosts is typically acute and cytopathic. We are investigating the nature of this difference to identify responsible host and viral factors. We previously published a study comparing LGTV virus infection in mammalian and tick cell lines utilizing molecular virology as well as confocal microscopy, electron microscopy, and electron tomography. Flavivirus infection in mammalian cell lines is accompanied by massive proliferation and rearrangement of cellular membrane, derived mainly from endoplasmic reticulum. Electron tomography revealed virus-induced spherical vesicles thought to protect replicative intermediates from intracellular antiviral systems. In contrast to mammalian cells, TBFV-infection in tick cells shows delayed and decreased membrane proliferation. Additionally, electron tomography of infected tick cells shows a shift from spherical vesicles to tubular profiles, especially in the context of persistently infected cells. In 2015, we continued these structural studies to cultures of mouse primary embryonic brain. LGTV infection was largely restricted to cells of neuronal origin, but glial cells also were infected. Ultrastructural studies revealed results similar to those observed in mammalian cell lines, although the frequency of the tubular profiles appeared greater than in permanent cell lines. Cellular structures associated with viral replication and virions were observed all along the processes of neurons, suggesting that these components are transported to actual nerve endings. The viral proteins and cellular partners responsible for the membrane rearrangements are a topic of interest in BVBV. Therefore, we have developed molecular clones expressing the nonstructural proteins, either singly or in clusters, that should prove suitable for evaluating the role of specific proteins in the rearrangement of the cellular membranes. It is hoped that the vector backbones should allow for good expression in both mammalian and tick cell lines. We have determined that expression in mammalian cells is higher using vectors with the chicken beta-actin promoter, and that Effectene is most efficacious transfection reagent. Results implicate that NS4a in the ER expansion and membrane rearrangement associated with TBFV infection. These studies were put on hold in order to begin work on Zika virus. This previous year, we initiated similar studies with ZIKV in a human neuroblastoma cell line (SK-N-SH) and mosquito cell lines (Aedes aegypti CCL-125 and A. albopictus C6/36). ZIKV replicated well in SK-N-SH and C6/36, but failed to grow in CCL-125. ZIKV caused an acute lytic crisis accompanied by frank apoptosis in SK-N-SH cells, but the effects of infection in C6/36 cells were imperceptible despite the fact that activated caspase 3 staining was readily apparent. These findings mirror those we have seen with the TBFV. The results of ultrastructural studies on SK-N-SH and C6/36 cells revealed that the cytoarchitecture of ZIKV infection was similar to that seen previously with the TBFV. However, the application of dual-tilt electron tomography lead to resolution substantially better than our previous work. Molecular biology and molecular pathogenesis of acute and persistent VBFV infection. Persistent infection plays a crucial role in natural life cycle of TBFV in rodent and arthropod hosts, and may also be responsible for prolonged debilitating sequelae observed in survivors of acute TBFV infection. However, this aspect of TBFV biology has been little studied. Experimental work by others on MBFV and the recent findings that ZIKV can persist in human semen and other sites for extended periods of time strongly imply that persistence of MBFV may also be an underappreciated feature of those infections. In the past year, we extended our study of TBFV persistence to inspect the cellular transcriptome during the initiation and maintenance of persistent infection. As noted, LGTV infection causes an acute lytic crisis of most cells, and we have shown the mechanism of cell death is apoptosis. The surviving cells somehow evade apoptosis and go on to establish persistent infection. Our agnostic deep sequencing approach to the study of cellular transcriptomics confirmed that gene pathways associated with cell survival and apoptosis avoidance were involved. A signature of 451 genes was associated with the initiation of persistence. Intensive bioinformatics perusal using Ingenuity Pathway Analysis and other similar software packages, revealed that networks associated with cell survival were playing a role. The acute phase was associated with networks and pathways. We also used our novel deep sequencing approach to examine the viral and cellular transcriptome during TBFV infection in tick cells (ISE-6). ISE-6 cells develop a persistent infection with no apparent acute phase. Although the cellular transcriptome analysis is still in progress, there is no evidence of DI genomes at any point. This is a substantial difference from mammalian cells, and has been submitted for publication. In other studies, we have initiated studies to identify cellular partners involved in egress of TBFV from infected mammalian and tick cells. This work capitalizes on proteomic work done by a current post-doctoral fellow in the lab. Peromyscus leucopus is the identified reservoir species for that autochthonous pathogenic POWV. We have begun experiments to characterize POWV/DTV infection in the P. leucopus mice. Both LGTV and POWV are severely restricted for growth in commerci